Preparation of ROS-responsive hydrogel loaded with Cu5.4O nanoparticles and its application in the treatment of radiation-induced skin injury

Abstract

This invention relates to the field of biomedical technology and provides a Cu-loaded... 5.4 Preparation of ROS-responsive hydrogels composed of O nanoparticles and their application in the treatment of radiation-induced skin injuries. This invention prepares a hydrogel with microenvironment ROS responsiveness, self-healing properties, good tissue adhesion, and loaded with a strong antioxidant, Cu. 5.4 O-NPs hydrogels. This hydrogel achieves Cu through ROS-responsive cleavage of borate ester bonds. 5.4 The controlled, targeted release of O-NPs at the site of radiation-induced skin injury (RISI) improves the pathological microenvironment of RISI and accelerates wound healing. Experiments have confirmed the good biocompatibility of this hydrogel, and proteomic analysis has revealed the signaling pathways involved in its action, establishing a stable and reliable development plan and providing a theoretical and experimental foundation for subsequent industrialization. This invention provides a novel, highly effective, targeted material for the clinical prevention and treatment of RISI, with broad prospects for clinical translational applications.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to preparation of ROS-responsive hydrogel loaded with Cu 5.4 O nanoparticles and application thereof in treatment of radioactive skin injury. BACKGROUND

[0002] Radioactive skin injury (RISI) is a major adverse reaction caused by radiotherapy. Research data shows that the probability of RISI occurring in tumor patients during radiotherapy is as high as 91.4%, and the clinical manifestations are skin and mucosa erythema, desquamation, ulceration and even necrosis, which easily leads to interruption of radiotherapy and seriously affects the treatment effect. The pathogenesis is mainly oxidative stress injury of skin caused by radiation, however, there is still a lack of ideal prevention and treatment means in the clinic.

[0003] In the existing clinical treatment, there is no unified treatment gold standard for radioactive skin injury. The conventional treatment mostly uses external steroid hormone ointments such as mometasone furoate and betamethasone, but the actual treatment effect is poor, and is often accompanied by serious side effects such as hypertension and heart disease, which is difficult to meet the long-term treatment needs of patients. From the pathological mechanism, acute radioactive skin injury is mostly related to epidermal and dermal cell changes and inflammatory reactions; chronic injury is mainly affected by radiation on angiogenesis, and skin appendages and glands are also atrophied due to radiation damage. Therefore, there is an urgent need for a treatment method that can comprehensively repair the full layer of skin, covering the damage of skin appendages and glands.

[0004] In recent years, hydrogel materials have attracted much attention in the field of drug release. As a 3D hydrophilic polymer network formed by chemical and / or physical crosslinking in an aqueous solution, compared with traditional dressings, it has the advantages of moisturizing, good permeability, antibacterial, good biocompatibility, self-cleaning and the like. Especially the microenvironment-responsive hydrogel can respond to stimuli in the microenvironment such as pH, temperature, glucose and reactive oxygen species (ROS), so that the crosslinked network is broken in time to release the loaded drugs, realizing controllable and targeted local release of drugs and avoiding systemic toxicity. In addition, hydrogels themselves have biological functions such as anti-inflammatory and antioxidant, and are widely used in the field of tissue damage repair. Current research has confirmed that the increase of local ROS and RNS induced by radiation is the main cause of skin injury. As a safe nanoscale antioxidant, the new type of metal material Cu 5.4 O nanoparticle (Cu 5.4 O-NPs) has a relatively simple preparation process, can act as catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) analogues, and has broad-spectrum and high-efficiency ROS scavenging capacity. Compared with known nano-antioxidants, Cu 5.4O-NPs have stronger antioxidant capacity, and the working dose is 2-3 orders of magnitude lower. Moreover, Cu element has the function of promoting angiogenesis, can stimulate the expression of platelet-derived growth factor (PDGF), basic fibroblast growth factor (BFGF) and vascular endothelial growth factor (VEGF), and is beneficial to the growth of granulation tissue and tissue repair. However, its application is limited because its properties are difficult to act on the local injury for a long time.

[0005] At present, intelligent hydrogel (hydrogel nanocomposite) is gradually replacing traditional hydrogel and applied in the biomedical field, and its advantages are obvious: first, it has hydrophilicity and can produce synergistic effect with the characteristics of embedding nanoparticles; second, it has high drug loading capacity; third, by virtue of the hydrophilic shell, the drug can avoid the recognition of the lymphatic system in the blood circulation, realize long-time circulation, or be enriched in the lesion site through strong penetration and retention effect. Therefore, efficient loading and drug controlled release have become an important direction of the intelligent development of current hydrogel. In view of the problems of large dose, poor targeting and long time of RISI symptomatic drugs in current clinical treatment, and the curative effect of the composite hydrogel drug loading system in RISI treatment has not been studied, the present application proposes a Cu 5.4 Preparation of ROS-responsive hydrogel loaded with Cu SUMMARY

[0006] The purpose of the present application is to provide a ROS-responsive hydrogel loaded with Cu 5.4 O-NPs, and its application in the treatment of radioactive skin injury, aims to solve the problems raised in the above background art.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] The ROS-responsive hydrogel loaded with Cu 5.4 O-NPs, the hydrogel is formed into a double network structure by cross-linking polyvinyl alcohol, borax and gelatin, and loaded with Cu 5.4 O-NPs; the hydrogel realizes ROS responsiveness through reversible rupture of borate ester bond, and releases Cu 5.4 O-NPs, and has self-healing property and tissue adhesion.

[0009] The preparation method of the hydrogel described above comprises the following steps:

[0010] Synthesis of Cu 5.4 O-NPs: copper chloride and ascorbic acid are dissolved in pure water respectively, then the copper chloride solution is placed in a volumetric flask, stirred under oil bath condition, and then the ascorbic acid solution is slowly added dropwise, and the stirring reaction is continued. After the reaction is completed, the reaction liquid is dialyzed, and finally the product is collected by centrifugation, that is, Cu 5.4 O-NPs.

[0011] Preparation of hydrogel: Polyvinyl alcohol powder is mixed with distilled water and heated in a water bath to fully dissolve it and form a homogeneous polyvinyl alcohol solution. Borax solution is added to the solution, followed by gelatin solution. The mixture is heated and stirred to obtain hydrogel. After sonication, it is transferred to an acrylic glass mold.

[0012] According to the above-described application of the hydrogel in the preparation of drugs for treating radiation-induced skin damage, the hydrogel accelerates wound healing by scavenging ROS, reducing inflammation, promoting cell migration and angiogenesis.

[0013] Furthermore, the hydrogel promotes the repair of radiation-induced skin damage by regulating the Rap1, NF-κB, and HIF-1 signaling pathways.

[0014] Based on the above-described application of hydrogels in the preparation of antioxidant or anti-inflammatory drugs, the hydrogels exert antioxidant and anti-inflammatory effects by scavenging ROS and inhibiting inflammatory factors.

[0015] A dressing containing the hydrogel described above, wherein the dressing is applied to the wound to achieve Cu 5.4 Targeted release of O-NPs and wound repair.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention is the first to combine ROS-responsive hydrogels with the strong antioxidant Cu. 5.4 By combining O-NPs, a hydrogel with microenvironment ROS responsiveness (based on the reversible breaking of borate ester bonds in the hydrogel structure), self-healing properties, good tissue adhesion, and loading of a strong antioxidant Cu was designed and prepared. 5.4 O-NPs hydrogel. This hydrogel achieves Cu through its ROS-responsive functionality. 5.4 The controlled, targeted release of O-NPs at the site of injury improves the pathological microenvironment of RISI and accelerates wound healing, overcoming the shortcomings of current clinical RISI symptomatic drugs, such as large dosage, poor targeting, and long treatment duration. In vitro and in vivo experiments have verified its good biocompatibility, and proteomic analysis revealed that its action involves the Rap1, NF-κB, and HIF-1 signaling pathways, forming a stable and reliable development plan and providing a theoretical and experimental foundation for subsequent industrialization. This invention provides new materials and methods for the prevention and treatment of RISI in clinical radiotherapy patients, helping to improve patient compliance, reduce cancer recurrence rates, and has broad prospects for clinical translational applications. Attached Figure Description

[0018] Figure 1 To successfully synthesize Cu 5.4O@PVA / B / Gel hydrogel; where A represents Cu observed by transmission electron microscopy (TEM). 5.4 O-NPs particle size, morphology, and uniformity; B represents Cu. 5.4 Quantitative analysis of O-NPs particle size; C represents the particle size of the prepared Cu using a UV spectrophotometer. 5.4 O-NPs samples were analyzed; D is the X-ray photoelectron spectrum of C1s; E is the X-ray photoelectron spectrum of Cu2p; F is the X-ray photoelectron spectrum of O1s; G is the synthesized Cu 5.4 Schematic diagram of O@PVA / B / Gel hydrogel; H represents the morphological characteristics and pore size of PVA / B / Gel hydrogel observed by scanning electron microscopy (SEM); I represents the Cu observed by scanning electron microscopy (SEM). 5.4 Morphological characteristics and pore size of O@PVA / B / Gel hydrogel.

[0019] Figure 2 Cu 5.4 Properties of O@PVA / B / Gel hydrogel; where A is Cu 5.4 Adhesion strength of O@PVA / B / Gel hydrogel on different materials; B represents Cu. 5.4 The freeformability of O@PVA / B / Gel hydrogel; C is Cu 5.4 Adhesion and conformability of O@PVA / B / Gel hydrogel; D is Cu 5.4 The self-healing properties of O@PVA / B / Gel hydrogel; E represents Cu. 5.4 Swelling properties of O@PVA / B / Gel hydrogel; F represents Cu 5.4 The scavenging ability of O@PVA / B / Gel hydrogel for DPPH, O2⁻, and OH⁻ free radicals; G represents Cu. 5.4 Drug release capacity of O@PVA / B / Gel hydrogel under different concentrations of H2O2.

[0020] Figure 3 Cu 5.4 The O@PVA / B / Gel hydrogel exhibited excellent biocompatibility both in vitro and in vivo. Image A shows the survival / death staining of HUVEC cells after 1, 2, and 3 days of treatment; image B shows mouse cells injected via tail vein with saline and Cu, respectively. 5.4 O-NPs or Cu 5.4 H&E staining images of major organs (heart, liver, spleen, lung, kidney) after 24 hours of O@PVA / B / Gel hydrogel; C shows HaCaT cells with different concentrations of Cu. 5.4Cell viability after O-NPs treatment; DG represents serum liver and kidney function-related biochemical indicators, including blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (ALT), and aspartate aminotransferase (AST); H represents different concentrations of Cu. 5.4 Hemolysis rate of erythrocytes after O-NPs treatment; data are expressed as mean ± standard deviation (mean ± SD). Statistical significance: * p <0.05;** p <0.01; *** p <0.001; **** p <0.0001; ns is not significant.

[0021] Figure 4 Cu 5.4 O-NPs promote cell migration, inhibit apoptosis, reduce inflammation, and scavenge ROS; among them, A represents the ability of different concentrations of Cu observed using confocal microscopy. 5.4 The effect of O-NPs on the ROS scavenging ability of HUVECs after irradiation; B shows the levels of HUVEC-related proteins CAT, 4-HNE, TNF-α, TGF-β, and Cleaved Caspase-3 detected by Western blot; C shows the detection of Cu by flow cytometry. 5.4 The effects of O-NPs on radiation-induced apoptosis and necrosis of HUVEC cells; D represents the quantitative analysis of different concentrations of Cu using a fluorescence microplate reader. 5.4 O-NPs' ability to scavenge ROS in HUVECs; E represents the statistical analysis of the ratio of necrotic to apoptotic cells in HUVECs under flow cytometry; F represents the effect of Cu (80-320 ng / mL) on reducing radiation damage to HUVECs 72 h after irradiation, as analyzed by CCK-8 assay; G represents the statistical analysis of HUVEC-related protein CAT detected by Western blot; H represents the statistical analysis of HUVEC-related protein Cleaved Caspase-3 detected by Western blot; I represents the statistical analysis of HUVEC-related protein 4-HNE detected by Western blot; J represents the statistical analysis of HUVEC-related protein TNF-α detected by Western blot; K represents the statistical analysis of HUVEC-related protein TGF-β detected by Western blot. p <0.05;** p <0.01; *** p <0.001; **** p <0.0001; ns is not significant.

[0022] Figure 5 Cu 5.4 O-NPs promote cell migration; where A represents different concentrations of Cu. 5.4The effect of O-NPs on Hacat migration at 0h, 12h, and 24h after irradiation; B represents different concentrations of Cu. 5.4 The effect of O-NPs on HUVEC migration at 0h, 12h, and 24h after irradiation; C represents different concentrations of Cu. 5.4 Transwell experimental results of O-NPs on HUVEC and Hacat after irradiation; D represents different concentrations of Cu. 5.4 Statistical analysis of O-NPs on the Hacat migration healing area after irradiation; E represents different concentrations of Cu. 5.4 Statistical analysis of O-NPs on the migration and healing area of ​​HUVECs after irradiation; F represents different concentrations of Cu. 5.4 Statistical analysis of the number of HUVECs migrating after irradiation using O-NPs; G represents different concentrations of Cu. 5.4 Statistical analysis of the number of Hacat migrations after irradiation using O-NPs. p <0.05;** p <0.01; *** p <0.001; **** p <0.0001; ns is not significant.

[0023] Figure 6 Cu 5.4 O@PVA / B / Gel hydrogel promotes the healing of RISI; where A is the X-ray linear accelerator and wound localization map for establishing a mouse skin injury model; B is representative photographs and relative wound areas of RISI after different treatments on days 0, 3, 7, 10, and 14 of the established mouse skin injury model; C is the thickness of newly formed epidermis, granulation tissue thickness, and number of newly formed capillaries in different treatment groups on day 14, assessed by HE staining; D is the deposition of collagen fibers in the dermis on day 14, assessed by Masson staining; E is the expression of CD31 and α-SMA in wound tissue on days 7 and 14, detected by immunofluorescence staining; F is the statistical analysis of the relative wound healing area in different treatment groups within 14 days; G is the statistical analysis of the number of angiogenesis in different treatment groups on day 14; H is the statistical analysis of the volume fraction of collagen fibers in different treatment groups on day 14; I is the statistical analysis of the number of CD31(+) vessels in different treatment groups on days 7 and 14.

[0024] Figure 7 Cu 5.4 Proteomic information on the regulation of RISI repair and healing by O@PVA / B / Gel hydrogel; where A represents the Normol group, Control group, and Cu group. 5.4 Hierarchical clustering dendrogram of differentially expressed proteins in the periwound tissue of the O@PVA / B / Gel group; B represents principal component analysis of the periwound tissue of different treatment groups; C represents the Control group and Cu group. 5.4Volcanic plot analysis of O@PVA / B / Gel group; D represents the Control group and Cu. 5.4 Radar plot of the top 30 differentially expressed proteins in the O@PVA / B / Gel group; E is an enrichment bubble plot showing the 20 most significantly enriched functions; F is an enrichment bubble plot of the Control group and Cu 5.4 KEGG enrichment analysis of differentially expressed proteins in the O@PVA / B / Gel group. Detailed Implementation

[0025] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0026] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Successful preparation of multifunctional Cu 5.4 O@PVA / B / Gel hydrogel;

[0028] Synthesis of Cu 5.4 O-NPs: A simple and economical method for preparing Cu 5.4 O-NPs. The simplified steps are as follows: Add 10mM copper chloride (CuCl2, providing Cu...) 2+ CuCl2 and 400 mM ascorbic acid (AA, reducing agent) were dissolved separately in 50 mL of pure water. Then, the CuCl2 solution was placed in a 250 mL volumetric flask and stirred in an 80°C oil bath. The AA solution was then slowly added dropwise, and the reaction was continued with stirring for 14–16 hours. After the reaction was complete, the reaction solution was dialyzed for 48 hours, and finally the product was collected by centrifugation to obtain CuCl2. 5.4 O-NPs.

[0029] Preparation of Cu 5.4 O@PVA / B / Gel hydrogel: 4g of polyvinyl alcohol (PVA) powder was mixed with 23ml of distilled water (16wt%) and heated in a water bath at 98°C for 2 hours to fully dissolve and form a homogeneous PVA solution. 4ml of borax (B) solution (0.005mmol / L) was added to the solution, followed by 4ml of gelatin (Gel) solution (0.08mmol / L). The mixture was heated at 98°C and stirred thoroughly for 1 hour to obtain Cu. 5.4 O@PVA / B / Gel hydrogel, sonicated for 30 minutes to remove air bubbles, then transferred to an acrylic glass mold.

[0030] 1.1 Cu 5.4 Characterization of O@PVA / B / Gel hydrogel;

[0031] The prepared Cu was observed by transmission electron microscopy (TEM). 5.4 O-NPs, from Figure 1 As seen in samples A and B, the particle size is relatively uniform, with a diameter of 4-5 nm, and an average diameter of 4.0-5.0 nm in the dry state. Cu was analyzed using a UV spectrophotometer. 5.4 O-NPs samples, from Figure 1 As can be seen from the C, its absorption intensity gradually increases with increasing wavelength, with an absorption peak at 600 nm, after which it gradually decreases with further increasing wavelength. The freeze-dried Cu... 5.4 X-ray photoelectron spectroscopy (XPS) analysis of O-NPs powder ( Figure 1 (D, E, and F). Figure 1 The C 1s spectrum of D showed a single symmetrical peak at 284.8 eV, which was attributed to the C–C / C–H bonds in the polyvinyl alcohol, gelatin and borax network, indicating that the organic skeleton remained stable. Figure 1 The middle E shows that Cu 5.4 O-NPs show only two strong peaks at 932.6 eV and 953.0 eV before and after oxidation, which is attributed to Cu 2p 2 / 3 and Cu 2p 1 / 2 The binding energy indicates that the reaction did not produce pure copper nanoparticles, but rather Cu. 0 and Cu + A mixture of Cu and Cu2O-NPs. Figure 1 The broad-scan spectrum of the medium-F further verified the presence of C 1s (~285 eV), O 1s (~531 eV), and Cu 2p (~933 eV) signals in the material, with no impurity peaks observed, proving that Cu 5.4 O nanoparticles were uniformly dispersed and successfully embedded in the composite hydrogel. Therefore, the resulting ultrasmall copper-based nanoparticles are represented as Cu. 5.4 O-NPs. Cu was loaded by crosslinking polyvinyl alcohol with borax and gelatin. 5.4 O-NPs were used to prepare a dual-network multifunctional hydrogel Cu. 5.4 O@PVA / B / Gel ( Figure 1 (G). SEM observation revealed that the hydrogel exhibited a porous and irregular network structure, and Cu was introduced. 5.4 O-NPs did not cause significant changes in the micromorphology and structure of the PVA / B / Gel gel. Figure 1 (H and I in the middle).

[0032] 1.2 Cu 5.4 Performance of O@PVA / B / Gel hydrogel;

[0033] Synthetic Cu 5.4The O@PVA / B / Gel hydrogel has a soft and elastic texture. Adhesion strength tests showed that it can adhere firmly to various material surfaces and remains intact and tightly bonded to the finger even after bending and twisting. Figure 2 (A and C in the middle). This hydrogel possesses freeform molding and shape memory properties, allowing it to be molded into a wide variety of 3D shapes. Figure 2 (Middle B). For example Figure 2 As shown in D, Cu 5.4 When an O@PVA / B / Gel hydrogel is cut in half lengthwise, with the cut surfaces of the two small pieces touching, and left for 30 seconds, the two pieces gradually fuse together until the cut surfaces disappear. This indicates that Cu... 5.4 The O@PVA / B / Gel hydrogel exhibits good self-healing properties. Furthermore, Cu was tested. 5.4 The swelling capacity of O@PVA / B / Gel hydrogel ( Figure 2 In the middle E), each group of hydrogels expanded rapidly in the tens of minutes before reaching equilibrium. Water molecules entered the hydrogel and interacted with the macromolecules inside the hydrogel. The swelling rate increased slowly and finally reached a stable state at about 215 minutes.

[0034] In the study of the ability to scavenge reactive oxygen species (ROS), three representative ROS—DPPH, O2·-, and OH·-—were selected. Figure 2 (F). The results show that Cu 5.4 The O@PVA / B / Gel hydrogel exhibits strong ROS scavenging ability, which is concentration-dependent. Its maximum scavenging rates for DPPH free radicals are 69% and 48%, respectively, equivalent to a cumulative release of approximately 220 ng / mL and 153 ng / mL of Cu, respectively. 5.4 O-NPs; for O2· - The maximum clearance rates were 54% and 37.9%, respectively, equivalent to a cumulative release of approximately 172 ng / mL and 121 ng / mL of Cu. 5.4 O-NPs; for OH· - The maximum clearance rates were 99.5% and 69.6%, respectively, equivalent to a cumulative release of approximately 318 ng / mL and 222 ng / mL of Cu. 5.4 O-NPs. This shows that Cu 5.4 O@PVA / B / Gel gel has a broad spectrum and strong ROS scavenging ability.

[0035] Cu 5.4 Cumulative copper release curves of O@PVA / B / Gel hydrogel under PBS, 400 μM and 800 μM H2O2 conditions ( Figure 2(G). Reversible cleavage of boronic acid ester bonds in the hydrogel structure was induced by adding H2O2 solutions of different concentrations: the ROS released by H2O2 contains diol groups, which competitively react with the diols in the boronic acid ester bonds, prompting the timely cleavage of the cross-linked network and releasing Cu. 5.4 O-NPs. Cu was detectable within 3 hours of incubation. 5.4 O-NPs are released from the hydrogel network. This key technology will provide an important guarantee for subsequent verification of the efficacy of ROS-responsive nanocomposite hydrogel drug delivery systems in preventing and treating RISI.

[0036] Example 2: Cu 5.4 O@PVA / B / Gel hydrogel has safe biocompatibility in vivo and in vitro;

[0037] Live / dead cell staining results showed ( Figure 3 In both control and blank control (IR) studies, HUVEC cells maintained good survival rates after 1-3 days of co-culture with hydrogel extract, showing no significant cytotoxicity compared to PBS (Control) and Cu loading. 5.4 The O-NPs hydrogel group showed higher cell viability. Further detection of free Cu... 5.4 The concentration-dependent toxicity of O-NPs in the range of 40-1280 ng / mL showed that, compared with the control, HUVEC cells had no significant toxicity at concentrations of 1280 ng / mL and below. Figure 3 (C). Blood compatibility testing showed ( Figure 3 (in H), in Cu 5.4 Within the concentration range of 80-1280 ng / mL for O-NPs, the hemolysis rate of erythrocytes was less than 5%, meeting the ISO 10993-4 standard for blood contact materials. In in vivo safety experiments (… Figure 3 (B) , 16 μg / kg free Cu 5.4 O-NPs or Cu 5.4 O@PVA / B / Gel hydrogel was injected into BALB / c mice via a single tail vein injection. Twenty-four hours later, major organs such as the heart, liver, spleen, lungs, and kidneys were harvested for H&E staining. Results showed that the tissue structures in each group were intact, without necrosis, hemorrhage, or significant inflammatory cell infiltration. Serum biochemical indicators (BUN, CRE, ALT, AST) were normal. Figure 3 (DG), with no liver or kidney function damage. In summary, Cu 5.4 O-NPs and Cu 5.4 O@PVA / B / Gel exhibited excellent in vivo and in vitro biocompatibility and systemic tolerance under acute exposure conditions.

[0038] Example 3: Cu 5.4O-NPs have the abilities of promoting cell migration, anti-cell apoptosis, anti-inflammation and scavenging ROS;

[0039] 3.1 Cu 5.4 O-NPs have the abilities of anti-cell apoptosis, anti-inflammation and scavenging ROS in vitro;

[0040] Using human umbilical vein endothelial cells (HUVEC) to detect the protective effect of Cu 5.4 O-NPs on ROS-damaged cells in vitro. HUVEC cells were irradiated with 18 Gy of X linear accelerator (IR) and cultured for 72 h. Confocal microscopy observation ( Figure 4 in A) showed that the intracellular ROS level (DCFH-DA) in the IR group was significantly increased, while in the cells pretreated with Cu 5.4 O-NPs, with the increase of the concentration of Cu 5.4 O-NPs, the fluorescence intensity gradually decreased, indicating that the ROS level was significantly reduced. The quantitative analysis of intracellular ROS level by fluorescence microplate reader ( Figure 4 in D) further confirmed this result. In addition, through Western blot detection ( Figure 4 in B), it was found that compared with the IR group, the level of catalase (CAT), an antioxidant protease, was significantly increased in the Cu (160 ng / mL) group ( Figure 4 in G) ( p < 0.01); compared with the IR group, the level of 4-hydroxynonenal (4-HNE), an index of oxidative stress injury, was significantly reduced in the Cu (320 ng / mL) group ( Figure 4 in I) ( p < 0.001), indicating that Cu 5.4 O-NPs have significant antioxidant ability and can reduce radiation-induced oxidative stress injury by increasing the expression level of antioxidant protease and decreasing the expression level of oxidative stress injury index. Further, through flow cytometry ( Figure 4 in C) to detect the effect of Cu 5.4 O-NPs on radiation-induced cell apoptosis and necrosis, the results showed that Cu 5.4 O-NPs significantly reduced the proportion of radiation-induced cell apoptosis and necrosis ( Figure 4 in E), and Western blot detection showed that Cleaved Caspase-3 involved in apoptosis execution was significantly lower in the Cu (160 - 320 ng / mL) group than in other groups ( Figure 4 in H) ( p < <0.01), further proving the ability of Cu 5.4 O-NPs to scavenge ROS and protect cells at the cellular level. The results of CCK-8 analysis ( Figure 4The study also showed that Cu (80-320 ng / mL) could alleviate radiation-induced cell damage. Western blot analysis of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) revealed that TNF-α promotes inflammatory responses, while TGF-β is involved in cell growth, differentiation, development, and wound repair. The results showed that compared to the IR group, TNF-α was significantly reduced in the Cu (320 ng / mL) group. Figure 4 J, p <0.01), indicating that Cu 5.4 O-NPs exhibited significant anti-inflammatory effects; compared to the IR group, TGF-β was significantly reduced in the Cu (320 ng / mL) group. Figure 5 Zhong K, p <0.001), indicating Cu 5.4 O-NPs possess significant abilities to promote HUVEC growth, differentiation, and repair. These results indicate that Cu... 5.4 O-NPs exhibit excellent abilities to promote cell migration, inhibit apoptosis, reduce inflammation, and scavenge ROS.

[0041] 3.2 Cu 5.4 O-NPs promote cell migration;

[0042] Detection of Cu using human epidermal keratinocytes (Hacat) and human umbilical vein endothelial cells (HUVEC) 5.4 O-NPs enhanced migration ability in vitro. HaCaT cells and HUVEC cells were irradiated with 8 Gy and 18 Gy X-ray linear accelerators, respectively. Cell scratch assays showed that, 12 h and 24 h after irradiation and subsequent drug intervention, compared with the IR group, HaCaT cells in the Cu (160-320 ng / mL) group ( Figure 5 (A and D) and HUVEC cells ( Figure 5 The migration rates of both B and E were significantly enhanced. p <0.001). The Transwell assay results were similar to the scratch assay; compared to the IR group, the Cu (160-320 ng / mL) group showed a significant increase in the number of migrating HaCaT and HUVEC cells per well. p <0.001) Figure 6 (C, F, and G). The above results indicate that Cu 5.4 O-NPs have a strong ability to promote cell migration.

[0043] Example 4: Cu 5.4 O@PVA / B / Gel hydrogel promotes the healing of RISI;

[0044] To investigate Cu 5.4The O@PVA / B / Gel hydrogel demonstrated its ability to repair RISI, and a model of X-ray linear accelerator-induced radiation-induced skin injury was established. Figure 6 (A) Eight-week-old male BALB / c mice were selected. A circular locating point with a diameter of 1 cm was made on the left buttock of the mouse. The mouse was first irradiated once with 15 Gy using an X-ray linear accelerator, and the skin tissue at this location was immediately excised after irradiation. The mice were divided into groups of 8: IR+Control group: no postoperative treatment; IR+PVA / B / Gel group: PVA / B / Gel hydrogel was applied to the wound after surgery; IR+Cu 5.4 O-NPs group: Mice were given Cu at the wound site after surgery. 5.4 O-NPs (125 ng / kg); IR+Cu 5.4 O@PVA / B / Gel group: Postoperative Cu 5.4 O@PVA / B / Gel hydrogel was applied to the wound site of mice. ROS and inflammatory responses at the wound site affect the speed of wound healing, such as... Figure 6 As shown in Figure B, on postoperative days 3, 7, 10, and 14, IR+Cu 5.4 Mice in the O@PVA / B / Gel group showed significantly better wound healing than those in the IR+Control group, demonstrating significantly accelerated wound healing. Fourteen days post-surgery, the IR+Cu group... 5.4 Mice in the O@PVA / B / Gel group had wounds that were nearly healed and moist, with a relative wound area of ​​only 4.75%, while mice in the IR+Control group had larger scabs on their wounds, with a relative wound area of ​​24.39%. Figure 6 (F in the middle) indicates that Cu 5.4 O@PVA / B / Gel hydrogel can promote wound repair and healing. Epidermal necrosis and collagen proliferation are typical pathological features of RISI. HE staining results show ( Figure 6 On day 14 after irradiation (C), epidermal necrosis was still present in the IR+Control group, while in the IR+Cu group... 5.4 The thickness of newly formed epidermis, the thickness of granulation tissue, and the number of newly formed capillaries in the O@PVA / B / Gel group (black arrows) were all significantly greater than those in the IR+Control group. Figure 6 (G), further confirming Cu 5.4 O@PVA / B / Gel hydrogel can promote wound healing. Masson staining shows (…). Figure 6 In the D and H groups, the IR+Control group showed less collagen fiber deposition, lighter color, and sparser arrangement in the wound tissue. IR+Cu 5.4 The O@PVA / B / Gel group showed coarser collagen fibers arranged in a woven mesh, similar to normal skin tissue, indicating that Cu... 5.4O@PVA / B / Gel hydrogel exhibits the ability to promote extracellular matrix (ECM) remodeling. Further immunofluorescence staining of wound tissue revealed the expression of CD31 and α-SMA, and the presence of IR+Cu... 5.4 The significantly enhanced CD31 and α-SMA fluorescence in the O@PVA / B / Gel group indicates that Cu 5.4 O@PVA / B / Gel hydrogel promotes the formation of new blood vessels ( Figure 7 (E and I).

[0045] Example 5: Cu 5.4 Proteomics analysis of O@PVA / B / Gel hydrogel repairing radiation-damaged skin tissue;

[0046] To clarify Cu 5.4 The mechanism of O@PVA / B / Gel hydrogel in treating radiation-induced skin injury was investigated, and proteomic analysis was performed on mice with radiation-induced skin injury treated with this hydrogel. A hierarchical clustering dendrogram was constructed using differentially expressed proteins in the surrounding tissue. Figure 7 (A) shows significant differences in protein expression among the three groups of mice, with red representing high expression and blue representing low expression. Principal component analysis showed ( Figure 7 (B), Normol group, Control group (IR_Control) and Cu 5.4 The O@PVA / B / Gel group (IR_Cu) exhibited significantly different gene expression patterns. (Volcano diagram) Figure 7 (C) Presents the Control group and Cu 5.4 Of the differentially expressed proteins O@PVA / B / Gel, 1240 were upregulated and 436 were downregulated out of 1676 significantly differentially expressed genes. A radar chart was created for the top 30 differentially expressed proteins. Figure 7 The middle circle (D) shows the relative expression levels, and the third circle shows the average quantitative levels of the two groups. Differentially expressed proteins with high expression levels will appear as peaks in the graph. For the Control group and Cu... 5.4 Enrichment analysis was performed on differentially expressed proteins in the O@PVA / B / Gel group, and the enrichment bubble diagram was generated. Figure 7 The study (E) revealed the 20 most significantly enriched functions, with significant differences observed in the regulation of acute inflammatory responses (highlighted in red). The study compared the Control and Cu groups... 5.4 KEGG enrichment analysis was performed on differentially expressed proteins in the O@PVA / B / Gel group. ​ By comparing and integrating differentially expressed genes with related pathways, significant signaling pathways were screened, suggesting that the Rap1, NF-κB, and HIF-1 pathways may be involved in Cu 5.4 O@PVA / B / Gel hydrogel promotes the repair and healing process of RISI.

[0047] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A Cu-supported 5.4 The application of ROS-responsive hydrogels composed of O nanoparticles in the preparation of drugs for treating radiation-induced skin damage, characterized in that... Specifically, the hydrogel accelerates wound healing by scavenging ROS, reducing inflammation, promoting cell migration and angiogenesis; The hydrogel is composed of a dual-network structure formed by cross-linking polyvinyl alcohol, borax, and gelatin, and is loaded with Cu. 5.4 O-NPs; the hydrogel achieves ROS responsiveness through the reversible breaking of borate ester bonds, releasing Cu in the ROS microenvironment. 5.4 O-NPs, and have self-healing properties and tissue adhesiveness; The method for preparing the hydrogel includes the following steps: Synthesis of Cu 5.4 O-NPs: Copper chloride and ascorbic acid were dissolved separately in pure water. The copper chloride solution was then placed in a volumetric flask and stirred in an oil bath. Ascorbic acid solution was then slowly added dropwise while stirring continuously. After the reaction was complete, the reaction solution was dialyzed, and the product was collected by centrifugation to obtain Cu. 5.4 O-NPs; Preparation of hydrogel: Polyvinyl alcohol powder is mixed with distilled water and heated in a water bath to fully dissolve it and form a homogeneous polyvinyl alcohol solution. Borax solution is added to the solution, followed by gelatin solution. The mixture is heated and stirred to obtain hydrogel. After sonication, it is transferred to an acrylic glass mold.

2. A Cu-supported 5.4 The application of ROS-responsive hydrogels composed of O nanoparticles in the preparation of antioxidant or anti-inflammatory drugs is characterized by, Specifically, the hydrogel exerts its antioxidant and anti-inflammatory effects by scavenging ROS and inhibiting inflammatory factors.

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